膜蕨科植物rbcL基因的适应性进化和共进化分析

膜蕨科植物是薄囊蕨类中种类最多的科,主要分布在潮湿的热带地区,拥有陆生、附生、半附生和攀生等多种生态型。为进一步了解膜蕨科植物辐射式物种分化的分子适应机制,该研究在时间框架下采用位点模型对膜蕨科植物rbc L基因的进化式样进行分析。结果表明:共鉴定出6个氨基酸正选择位点(125I、227L、231A、258F、304S和351L),其中位点304S位于环六上,对维持Rubisco功能有重要作用。此外,还计算了Rubisco大亚基内部氨基酸位点之间的共进化关系,共检测出39组(35个氨基酸)共进化位点,其中位点在α螺旋上的占46%,在β折叠上的占14%。膜蕨科植物rbc L基因这种复杂的进化式样可能与其起源较早有关。鉴于此,基于UCLD分子钟模型对膜蕨科植物的分化时间进行了估计,结果显示膜蕨科植物首次发生分歧的时间在三叠纪早期,瓶蕨属和膜蕨属的分歧时间分别发生在侏罗纪早期和白垩纪晚期,并且得出陆生生态型是其它生态型进化的基础,推测最近几次最热事件可能对物种分化的形成产生一定的作用。该研究结果对认识膜蕨科植物如何应对被子植物兴起所导致的陆地生态系统改变具重要意义。     

Comparative genomics and bioenergetics.

Bacterial and archaeal complete genome sequences have been obtained from a wide range of evolutionary lines, which allows some general conclusions about the phylogenetic distribution and evolution of bioenergetic pathways to be drawn. In particular, I searched in the complete genomes for key enzymes involved in aerobic and anaerobic respiratory pathways and in photosynthesis, and mapped them into an rRNA tree of sequenced species. The phylogenetic distribution of these enzymes is very irregular, and clearly shows the diverse strategies of energy conservation used by prokaryotes. In addition, a thorough phylogenetic analysis of other bioenergetic protein families of wide distribution reveals a complex evolutionary history for the respective genes. A parsimonious explanation for these complex phylogenetic patterns and for the irregular distribution of metabolic pathways is that the last common ancestor of Bacteria and Archaea contained several members of every gene family as a consequence of previous gene or genome duplications, while different patterns of gene loss occurred during the evolution of every gene family. This would imply that the last universal ancestor was a bioenergetically sophisticated organism. Finally, important steps that occurred during the evolution of energetic machineries, such as the early evolution of aerobic respiration and the acquisition of eukaryotic mitochondria from a proteobacterium ancestor, are supported by the analysis of the complete genome sequences.     

Immunolocalization and distribution of form ii rubisco in the pyrenoid and chloroplast stroma of amphidinium carterae and form i rubisco in the symbiont-derived plastids of peridinium foliaceum (dinophyceae)

Chloroplasts of peridinin-containing dinoflagellates have recently been shown to contain Form II Rubisco, which consists of large subunits only and is coded by nuclear genes. We have used immunoelectron microscopy to determine the distribution of Form II and Form I Rubisco in dinoflagellates. In sections of Amphidinium carterae Hulburt, the pyrenoid was intensely labeled and the rest of the chloroplast moderately labeled by antisera to Form II Rubisco from the purple non-sulfur bacterium Rhodospirillum rubrum and the symbiotic dinoflagellate Symbiodinium sp. No labeling was observed when sections were exposed to antiserum against Form I Rubisco of the haptophyte alga Isochrysis galbana. In contrast, cell sections of the dinoflagellate Peridinium foliaceum (Stein) Biecheler, whose chloroplasts belong to a diatom endosymbiont, showed no labeling with the two antisera against Form II Rubisco, but heavy pyrenoid labeling was present after treatment with antiserum against Form I Rubisco of I. galbana. The same immunolabeling results were obtained with the free-living diatom Phaeodactylum tricornutum Bohlin. Volumetric analysis of the distribution of Form II Rubisco in the chloroplast of A. carterae showed that, in cells grown under moderate photon irradiance, 72.9% of the plastid's Rubisco was localized in the pyrenoid, whereas in cells grown under low irradiance only 37.0% of the Rubisco was found in the pyrenoid. This light-induced concentration of Rubisco in the pyrenoid suggests that a CO₂–concentrating mechanism may elevate CO₂ within the pyrenoid, favoring the efficient fixation of CO₂ by pyrenoid Rubisco.     

The age of Rubisco: the evolution of oxygenic photosynthesis

The evolutionary history of oxygenesis is controversial. Form I of ribulose 1,5‐bisphosphate carboxylase/oxygenase (Rubisco) in oxygen‐tolerant organisms both enables them to carry out oxygenic extraction of carbon from air and enables the competitive process of photorespiration. Carbon isotopic evidence is presented from ~2.9 Ga stromatolites from Steep Rock, Ontario, Canada, ~2.9 Ga stromatolites from Mushandike, Zimbabwe, and ~2.7 Ga stromatolites in the Belingwe belt, Zimbabwe. The data imply that in all three localities the reef‐building autotrophs included organisms using Form I Rubisco. This inference, though not conclusive, is supported by other geochemical evidence that these stromatolites formed in oxic conditions. Collectively, the implication is that oxygenic photosynthesizers first appeared ~2.9 Ga ago, and were abundant 2.7–2.65 Ga ago. Rubisco specificity (its preference for CO₂ over O₂) and compensation constraints (the limits on carbon fixation) may explain the paradox that despite the inferred evolution of oxygenesis 2.9 Ga ago, the Late Archaean air was anoxic. The atmospheric CO₂:O₂ ratio, and hence greenhouse warming, may reflect Form I Rubisco's specificity for CO₂ over O₂. The system may be bistable under the warming Sun, with liquid oceans occurring in either anoxic (H₂O with abundant CH₄ plus CO₂) or oxic (H₂O with more abundant CO₂, but little CH₄) greenhouse states. Transition between the two states would involve catastrophic remaking of the biosphere. Build‐up of a very high atmospheric inventory of CO₂ in the 2.3 Ga glaciation may have allowed the atmosphere to move up the CO₂ compensation line to reach stability in an oxygen‐rich system. Since then, Form I Rubisco specificity and consequent compensation limits may have maintained the long‐term atmospheric disproportion between O₂ and CO₂, which is now close to both CO₂ and O₂ compensation barriers.     

Ribonucleotide reductases: divergent evolution of an ancient enzyme

Ribonucleotide reductases (RNRs) are uniquely responsible for converting nucleotides to deoxynucleotides in all dividing cells. The three known classes of RNRs operate through a free radical mechanism but differ in the way in which the protein radical is generated. Class I enzymes depend on oxygen for radical generation, class II uses adenosylcobalamin, and the anaerobic class III requires S-adenosylmethionine and an iron–sulfur cluster. Despite their metabolic prominence, the evolutionary origin and relationships between these enzymes remain elusive. This gap in RNR knowledge can, to a major extent, be attributed to the fact that different RNR classes exhibit greatly diverged polypeptide chains, rendering homology assessments inconclusive. Evolutionary studies of RNRs conducted until now have focused on comparison of the amino acid sequence of the proteins, without considering how they fold into space. The present study is an attempt to understand the evolutionary history of RNRs taking into account their three-dimensional structure. We first infer the structural alignment by superposing the equivalent stretches of the three-dimensional structures of representatives of each family. We then use the structural alignment to guide the alignment of all publicly available RNR sequences. Our results support the hypothesis that the three RNR classes diverged from a common ancestor currently represented by the anaerobic class III. Also, lateral transfer appears to have played a significant role in the evolution of this protein family.     

Evolution of RNA polymerases and branching patterns of the three major groups of archaebacteria

Summary The amino acid sequences of the largest subunits of the RNA polymerases I, II, and III from eukaryotes were compared with those of archaebacterial and eubacterial homologs, and their evolutionary relationships were analyzed in detail by a recently developed tree-making method, the likelihood method of protein phylogeny, as well as by the neighbor-joining method and the parsimony method, together with bootstrap analyses. It was shown that the best tree topologies predicted by the first two methods are identical, whereas the last one predicts a distinct tree. The maximum likelihood tree revealed that, after the separation from archaebacteria, the three eukaryotic RNA polymerases diverged from an ancestral precursor in the eukaryotic lineage. This result is contrasted with the published result showing multiple origins for the three eukaryotic polymerases. It was shown that eukaryotic RNA polymerase I evolved much more rapidly than RNA polymerases II and III: The N-terminal half of RNA polymerase I shows an extraordinarily high evolutionary rate, possibly due to relaxed functional constraints. In contrast the evolutionary rate of archaebacterial RNA polymerase is remarkably limited. In addition, including the second largest subunit of the RNA polymerase, a detailed analysis for the branching pattern of the three major groups of archaebacteria was carried out by the maximum likelihood method. It was shown that the three major groups of archaebacteria are likely to form a single cluster; that is, archaebacteria are likely to be monophyletic as originally proposed by Woese and his colleagues.     

The Monophyletic Origin of the Peridinin‐, and Fucoxanthin‐Containing Dinoflagellate Plastid Through Tertiary Replacement

The dinoflagellates contain diverse plastids of uncertain origin. To determine the origin of the peridinin‐ and fucoxanthin‐containing dinoflagellate plastid, we sequenced the plastid‐encoded psaA, psbA, and rbcL genes from various red and dinoflagellate algae. The psbA gene phylogeny, which was made from a dataset of 15 dinoflagellates, 22 rhodophytes, five cryptophytes, seven haptophytes, seven stramenopiles, two chlorophytes, and a glaucophyte as the outgroup, supports monophyly of the peridinin‐, and fucoxanthin‐containing dinoflagellates, as a sister group to the haptophytes. The monophyletic relationship with the haptophytes is recovered in the psbA + psaA phylogeny, with stronger support. The rubisco tree utilized the ‘Form I’ red algal type of rbcL and included fucoxanthin‐containing dinoflagellates. The dinoflagellate + haptophyte sister relationship is also recovered in this analysis. Peridinium foliaceum is shown to group with the diatoms in all the phylogenies. Based on our analyses of plastid sequences, we postulate that: (1) the plastid of peridinin‐, and fucoxanthin‐containing dinoflagellates originated from a common ancestor; (2) the ancestral dinoflagellate acquired its plastid from a haptophyte though a tertiary plastid replacement; (3) ‘Form II’ rubisco replaced the ancestral rbcL after the divergence of the peridinin‐, and fucoxanthin‐containing dinoflagellates; and (4) we confirm that the plastid of P. foliaceum originated from a Stramenopiles endosymbiont.     

Differences in carbon isotope discrimination of three variants of D-ribulose-1,5-bisphosphate carboxylase/oxygenase reflect differences in their catalytic mechanisms

The carboxylation kinetic (stable carbon) isotope effect was measured for purified d-ribulose-1,5-bisphosphate carboxylases/oxygenases (Rubiscos) with aqueous CO(2) as substrate by monitoring Rayleigh fractionation using membrane inlet mass spectrometry. This resulted in discriminations (Delta) of 27.4 +/- 0.9 per thousand for wild-type tobacco Rubisco, 22.2 +/- 2.1 per thousand for Rhodospirillum rubrum Rubisco, and 11.2 +/- 1.6 per thousand for a large subunit mutant of tobacco Rubisco in which Leu(335) is mutated to valine (L335V). These Delta values are consistent with the photosynthetic discrimination determined for wild-type tobacco and transplastomic tobacco lines that exclusively produce R. rubrum or L335V Rubisco. The Delta values are indicative of the potential evolutionary variability of Delta values for a range of Rubiscos from different species: Form I Rubisco from higher plants; prokaryotic Rubiscos, including Form II; and the L335V mutant. We explore the implications of these Delta values for the Rubisco catalytic mechanism and suggest that Rubiscos that are associated with a lower Delta value have a less product-like carboxylation transition state and/or allow a decarboxylation step that evolution has excluded in higher plants.     

Diversity and evolution of 5S rRNA gene family organization in Pythium

The 5S rRNA gene family organization among 87 species and varieties of Pythium was investigated to assess evolutionary stability of the two patterns detected and to determine which pattern is likely the ancestral state in the genus. Species with filamentous sporangia (Groups A-C according to the ITS phylogenetic tree for Pythium) had 5S genes linked to the rDNA repeat that were predominantly coded for on the DNA strand opposite to the one with the other rRNA genes (‘inverted’ orientation). A small group of species with contiguous sporangia (Group D) is related to Groups A-C but had unlinked 5S genes. The main group of species with spherical zoosporangia (Groups E-J) generally had unlinked 5S genes in tandem arrays. The six species in Group K, although they also have spherical sporangia, had linked genes on the same strand as the other rRNA genes ‘non-inverted’ and most of them had pairs of tandem 5S genes. The evolutionary stability of 5S sequence organization was compared with the stability of morphological characters as interpreted from a phylogeny based on ITS sequence analysis. Features of 5S sequence organization were found to be just as consistent within groups as were the morphological characters. To determine the ancestral type of 5S family organization, a survey of Phytophthora strains was conducted to supply an outgroup reference. The most parsimonious interpretation of the data in this survey yielded the tentative conclusion that the linked condition of the 5S sequences was ancestral.     

In Vivo Studies in Rhodospirillum rubrum Indicate That Ribulose-1,5-bisphosphate Carboxylase/Oxygenase (Rubisco) Catalyzes Two Obligatorily Required and Physiologically Significant Reactions for Distinct Carbon and Sulfur Metabolic Pathways

All organisms possess fundamental metabolic pathways to ensure that needed carbon and sulfur compounds are provided to the cell in the proper chemical form and oxidation state. For most organisms capable of using CO₂ as sole source of carbon, ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase (Rubisco) catalyzes primary carbon dioxide assimilation. In addition, sulfur salvage pathways are necessary to ensure that key sulfur-containing compounds are both available and, where necessary, detoxified in the cell. Using knock-out mutations and metabolomics in the bacterium Rhodospirillum rubrum, we show here that Rubisco concurrently catalyzes key and essential reactions for seemingly unrelated but physiologically essential central carbon and sulfur salvage metabolic pathways of the cell. In this study, complementation and mutagenesis studies indicated that representatives of all known extant functional Rubisco forms found in nature are capable of simultaneously catalyzing reactions required for both CO₂-dependent growth as well as growth using 5-methylthioadenosine as sole sulfur source under anaerobic photosynthetic conditions. Moreover, specific inactivation of the CO₂ fixation reaction did not affect the ability of Rubisco to support anaerobic 5-methylthioadenosine metabolism, suggesting that the active site of Rubisco has evolved to ensure that this enzyme maintains both key functions. Thus, despite the coevolution of both functions, the active site of this protein may be differentially modified to affect only one of its key functions.     

Early evolution of eukaryote feeding modes,cell structural diversity,and classification of the protozoan phyla Loukozoa,Sulcozoa, and Choanozoa

I discuss how different feeding modes and related cellular structures map onto the eukaryote evolutionary tree. Centrally important for understanding eukaryotic cell diversity are Loukozoa: ancestrally biciliate phagotrophic protozoa possessing a posterior cilium and ventral feeding groove into which ciliary currents direct prey. I revise their classification by including all anaerobic Metamonada as a subphylum and adding Tsukubamonas. Loukozoa, often with ciliary vanes, are probably ancestral to all protozoan phyla except Euglenozoa and Percolozoa and indirectly to kingdoms Animalia, Fungi, Plantae, and Chromista. I make a new protozoan phylum Sulcozoa comprising subphyla Apusozoa (Apusomonadida, Breviatea) and Varisulca (Diphyllatea; Planomonadida, Discocelida, Mantamonadida; Rigifilida). Understanding sulcozoan evolution clarifies the origins from them of opisthokonts (animals, fungi, Choanozoa) and Amoebozoa, and their evolutionary novelties; Sulcozoa and their descendants (collectively called podiates) arguably arose from Loukozoa by evolving posterior ciliary gliding and pseudopodia in their ventral groove. I explain subsequent independent cytoskeletal modifications, accompanying further shifts in feeding mode, that generated Amoebozoa, Choanozoa, and fungi. I revise classifications of Choanozoa, Conosa (Amoebozoa), and basal fungal phylum Archemycota. I use Choanozoa, Sulcozoa, Loukozoa, and Archemycota to emphasize the need for simply classifying ancestral (paraphyletic) groups and illustrate advantages of this for understanding step-wise phylogenetic advances.     

Post-genomic science: converting primary structure into physiological function

Exobiology, the study of the origin, evolution and distribution of life (including life on earth) within the context of cosmic evolution, is being given a remarkable boost by genome sequencing projects, which are now making the evolutionary histories of protein families routinely available. These histories comprise a multiple alignment for their protein sequences and the corresponding DNA sequences, an evolutionary tree showing the pedigree of these sequences, and reconstructed ancestral sequences for each node in the tree. In a post-genomic world having genomic sequences from an unlimited number of organisms, these histories will be used to connect structure, chemical reactivity, and physiological function to these families. This paper describes several “post-genomic” tools that exploit these evolutionary histories. They can be used to confirm or deny long distance homology between two protein families, identify proteins within a family that have new functions, and identify specific in vitro properties of the protein that are important for its physiological role. Evolution-based data structures for organizing large sequence databases are also described.     

Phylogenetic aspects of carbamoyl phosphate synthetase in lungfish: a transitional enzyme in transitional fishes

Carbamoyl phosphate synthetase (CPS) catalyses the formation of carbamoyl phosphate from glutamine or ammonia, bicarbonate and ATP. There are three different isoforms of CPS that play vital roles in two metabolic pathways, pyrimidine biosynthesis (CPS II) and arginine/urea biosynthesis (CPS I and CPS III). Gene duplication has been proposed as the evolutionary mechanism creating this gene family with CPS II likely giving rise to the CPS I/III clade. In the evolutionary history of this gene family it is still undetermined when CPS I diverged from CPS III on the path to terrestriality in the vertebrates. Transitional organisms such as lungfishes are of particular interest because they are capable of respiring via gills and with lungs and therefore can be found in both aquatic and terrestrial environments. Notably, enzymatic characterization of the mitochondrial CPS isoforms in this transitional group has not led to clear conclusions. In order to determine which CPS isoform is present in transitional animals, we examined partial sequences for liver CPS amplified from five species of lungfish, and a larger fragment of CPS from one lungfish species (Protopterus annectens) and compared them to CPS isoforms from other fish and mammals. Enzyme activities for P. annectens liver were also examined. While enzyme activities did not yield a clear distinction between isoforms (virtually equal activities were obtained for either CPS I or III), CPS sequences from the lungfishes formed a monophyletic clade within the CPS I clade and separate from the CPS III clade of other vertebrates. This finding implies that the mitochondrial isoform of CPS in lungfish is derived from CPS I and is likely to have a physiological function similar to CPS I. This finding is important because it supports the hypothesis that lungfish employ a urea cycle similar to terrestrial air-breathing vertebrates.     

凤尾蕨科植物rbcL基因的适应性进化分析

为深入理解蕨类植物辐射式物种分化的分子适应机制,在时间框架下,采用位点模型和分支-位点模型对凤尾蕨科植物rbcL基因的进化式样进行了分析.通过比较模型M1a/M2a和M7/M8,在氨基酸水平上共鉴定出6个正选择位点:1491、251M、255V、282F、359S和375F,其中位点282F对维持Rubisco功能有重要作用.分别检验凤尾蕨科的附生分支和水蕨类分支发现,前者不具适应性进化位点,而后者有两个位点(230A和247C)经历正选择.相对于荫蔽的光条件,水生生境可能对RbcL亚基的选择作用更强.另外,基于UCLD分子钟模型估算出的风尾蕨科各分支分化时间表明,该科物种丰富度的辐射式增长发生在新生代渐新世,推测古、始新世最热事件可能对物种分化的形成也产生一定作用.这对认识薄囊蕨类如何应对被子植物兴起导致的陆地生态系统改变具重要意义.     

Cell evolution and Earth history: stasis and revolution

This synthesis has three main parts. The first discusses the overall tree of life and nature of the last common ancestor (cenancestor). I emphasize key steps in cellular evolution important for ordering and timing the major evolutionary innovations in the history of the biosphere, explaining especially the origins of the eukaryote cell and of bacterial flagella and cell envelope novelties. Second, I map the tree onto the fossil record and discuss dates of key events and their biogeochemical impact. Finally, I present a broad synthesis, discussing evidence for a three-phase history of life. The first phase began perhaps ca 3.5 Gyr ago, when the origin of cells and anoxic photosynthesis generated the arguably most primitive prokaryote phylum, Chlorobacteria (= Chloroflexi), the first negibacteria with cells bounded by two acyl ester phospholipid membranes. After this 'chlorobacterial age' of benthic anaerobic evolution protected from UV radiation by mineral grains, two momentous quantum evolutionary episodes of cellular innovation and microbial radiation dramatically transformed the Earth's surface: the glycobacterial revolution initiated an oxygenic 'age of cyanobacteria' and, as the ozone layer grew, the rise of plankton; immensely later, probably as recently as ca 0.9 Gyr ago, the neomuran revolution ushered in the 'age of eukaryotes', Archaebacteria (arguably the youngest bacterial phylum), and morphological complexity. Diversification of glycobacteria ca 2.8 Gyr ago, predominantly inhabiting stratified benthic mats, I suggest caused serial depletion of 13C by ribulose 1,5-bis-phosphate caboxylase/oxygenase (Rubisco) to yield ultralight late Archaean organic carbon formerly attributed to methanogenesis plus methanotrophy. The late origin of archaebacterial methanogenesis ca 720 Myr ago perhaps triggered snowball Earth episodes by slight global warming increasing weathering and reducing CO2 levels, to yield runaway cooling; the origin of anaerobic methane oxidation ca 570 Myr ago reduced methane flux at source, stabilizing Phanerozoic climates. I argue that the major cellular innovations exhibit a pattern of quantum evolution followed by very rapid radiation and then substantial stasis, as described by Simpson. They yielded organisms that are a mosaic of extremely conservative and radically novel features, as characterized by De Beer's phrase 'mosaic evolution'. Evolution is not evenly paced and there are no real molecular clocks.     

Substitutions at methionine 295 of Archaeoglobus fulgidus ribulose-1,5-bisphosphate carboxylase/oxygenase affect oxygen binding and CO2/O2 specificity

Archaeoglobus fulgidus RbcL2, a form III ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), exhibits unique properties not found in other well studied form I and II Rubiscos, such as optimal activity from 83 to 93 degrees C and an extremely high kcat value (23 s-1). More interestingly, this protein is unusual in that exposure or assay in the presence of oxygen and high levels of CO2 resulted in substantial loss (85-90%) of activity compared with assays performed under strictly anaerobic conditions. Kinetic studies indicated that A. fulgidus RbcL2 possesses an unusually high affinity for oxygen (Ki=5 microM); O2 is a competitive inhibitor with respect to CO2, yet the high affinity for O2 presumably accounts for the inability of high levels of CO2 to prevent inhibition. Comparative bioinformatic analyses of available archaeal Rubisco sequences were conducted to provide clues as to why the RbcL2 protein might possess such a high affinity for oxygen. These analyses suggested the potential importance of several unique residues, as did additional analyses within the context of available form I-III Rubisco structures. One residue unique to archaeal proteins (Met-295) was of particular interest because of its proximity to known active-site residues. Recombinant M295D A. fulgidus Rubisco was less sensitive to oxygen compared with the wild-type enzyme. This residue, along with other potential changes in conserved residues of form III Rubiscos, may provide an understanding as to how Rubisco may have evolved to function in the presence of air.     

Techniques for determining protein sequence evolution applied to primates

Each amino acid in a protein is considered to be an individual, mutable characteristic of the species from which the protein is extracted. For a branching tree representing the evolutionary history of the known sequences in different species, our computer programs use majority logic and parsimony of mutations to determine the most likely ancestral amino acid for each position of the protein at each node of the tree. The number of mutations necessary between the ancestral and present species is summed for each branch and the entire tree. The programs then move branches to make many different configurations, from which we select the one with the minimum number of mutations as the most likely evolutionary history. We used this method to elucidate primate phylogeny from sequences of fibrinopeptides, carbonic anhydrase, and the hemoglobin beta, delta and alpha chains. All available sequences indicate that the early Pongidae had diverged into two lines before the divergence of an ancestor for the human line alone. We have constructed some probable ancestral sequences at major points during primate evolution and have developed tentative trees showing the order of divergences and evolutionary distances among primate groups. Further questions on primate evolution could be answered in the future by the detemination of the appropriate sequences.     

Changes in Rubisco kinetics during the evolution of C4 photosynthesis in Flaveria (Asteraceae) are associated with positive selection on genes encoding the enzyme

Rubisco, the primary photosynthetic carboxylase, evolved 3-4 billion years ago in an anaerobic, high CO(2) atmosphere. The combined effect of low CO(2) and high O(2) levels in the modern atmosphere, and the inability of Rubisco to distinguish completely between CO(2) and O(2), leads to the occurrence of an oxygenation reaction that reduces the efficiency of photosynthesis. Among land plants, C(4) photosynthesis largely solves this problem by facilitating a high CO(2)/O(2) ratio at the site of Rubisco that resembles the atmosphere in which the ancestral enzyme evolved. The prediction that such conditions favor Rubiscos with higher kcat(CO2) and lower CO(2)/O(2) specificity (S(C/O)) is well supported, but the structural basis for the differences between C(3) and C(4) Rubiscos is not clear. Flaveria (Asteraceae) includes C(3), C(3)-C(4) intermediate, and C(4) species with kinetically distinct Rubiscos, providing a powerful system in which to study the biochemical transition of Rubisco during the evolution from C(3) to C(4) photosynthesis. We analyzed the molecular evolution of chloroplast rbcL and nuclear rbcS genes encoding the large subunit (LSu) and small subunit (SSu) of Rubisco from 15 Flaveria species. We demonstrate positive selection on both subunits, although selection is much stronger on the LSu. In Flaveria, two positively selected LSu amino acid substitutions, M309I and D149A, distinguish C(4) Rubiscos from the ancestral C(3) species and statistically account for much of the kinetic difference between the two groups. However, although Flaveria lacks a characteristic "C(4)" SSu, our data suggest that specific residue substitutions in the SSu are correlated with the kinetic properties of Rubisco in this genus.     

Amplification of the ancient murine Lx family of long interspersed repeated DNA occurred during the murine radiation

We identified and characterized the relics of an ancient rodent Ll family, referred to as Lx, which was extensively amplified at the time of the murine radiation about 12 million years ago, and which we showed was ancestral to the modern L1 families in rat and mouse. Here we have extended our analysis of the Lx amplification by examining more murine and nonmurine species for Lx sequences using both blot hybridization and the polymerase chain reaction for a total of 36 species. In addition we have determined the relative copy number and sequence divergence, or age, of Lx elements in representative murine genera. Our results show that while Lx sequences are confined to murine genera, the extent of the amplification was different in the different murine lineages, indicating that the amplification of Lx did not precede, but was coincident with, the murine radiation. The implications of our findings for the evolutionary dynamics of L1 families and the utility of ancestral amplification events for systematics are discussed. Correspondence to: A.V. Furano